Cutaneous acariasis in the African clawed frog (Xenopus laevis)

Increased mortality was observed in a single colony of 50 Xenopus laevis. The frogs were used as oocyte donors in developmental biology studies. Necropsy findings included dermal erythema and petechiation consistent with red leg syndrome; dermal ulcerations and white, filamentous growths on the skin were consistent with Saprolegnia sp. Microscopic evaluation of the skin and fungus revealed an astigmatid mite similar to those of the genus Rhizoglyphus. The mite was also found in the water and the biological filter of the tanks housing the frogs. This mite is considered not to be a parasite of X. laevis; instead, it feeds off moss, fungi, and detritus. Subsequent evaluation of the sphagnum moss used for shipping the frogs from the supplier revealed the same mite in the moss. Our hypothesis is that the mite was introduced into the tank with the shipment of new frogs in sphagnum moss. The mites lived within the biological filter, and were only found after the growth of Saprolegnia sp. attracted the mites to the frogs. Laboratory animal care and veterinary personnel should consider non-pathogenic species of mites in the differential diagnosis of acariasis in Xenopus frogs.     

Nephroblastoma in the clawed frog Xenopus laevis.

One 2.5-3 year old female clawed frog (Xenopus laevis), out of a consignment of 4,000 frogs, was found to have an abnormal abdominal growth, weighing 7.9 g. The growth was examined histologically and on the basis of the abundant stroma and serially-arranged tubules nephroblastoma was diagnosed. The growth is not considered to be transmissible.     

Phenotypic flexibility in the intestinal enzymes of the African clawed frog Xenopus laevis

The intestinal plasticity of digestive enzymes of amphibian species is poorly known. The goal of this study was to characterize digestive enzyme profiles along the small intestine of adult frogs, Xenopus laevis, in response to an experimental diet. We acclimated adult X. laevis for 30 days either to carbohydrate-rich or protein-rich diets, and determined the morphology and digestive enzymes of the small intestine. We found a significant difference of aminopeptidase-N activity between carbohydrate-rich and protein-rich acclimated animals. We also found a little variation in the expression of maltase activity, which contrast with the proposed hypothesis about the existence of digestive tradeoff in vertebrates. This finding supports the adaptive modulation hypothesis and suggests that caution is called for when analyzing physiological data regarding assumed discrete trophic category of species.     

Factors affecting oogenesis in the South African clawed frog (Xenopus laevis)

Xenopus laevis, commonly known as the South African Clawed frog, is a hardy adaptable species that is relatively easy to maintain as a laboratory animal. Gametogenesis in wild Xenopus laevis is continuous and under ideal conditions, reproduction can occur year round. This unique aspect of amphibian reproduction offers an advantage over mammalian model systems: the eggs and oocytes collected from laboratory maintained Xenopus laevis provide an abundant and readily obtainable supply of material for cellular and biological research. However, many investigators report that laboratory Xenopus laevis go through periods of unexplained inefficient or complete failure of oocyte production or the production of poor quality oocytes. This results in experimental delays, inability to reproduce data, and ultimately the use of more animals. There is a lack of evidenced based information regarding the housing conditions that are necessary to optimize the health and fecundity of this species in captivity, but studies of wild Xenopus laevis have shown that temperature, age of the female, and nutrition are of key importance. The objective of this report is to review oogenesis with a special emphasis on these factors as they pertain to laboratory Xenopus laevis maintained for the purpose of providing a steady supply of eggs and oocytes. Harvesting methods and other experimental techniques that affect the quality of eggs and oocytes are also discussed.     

Artificial fertilization of gametes from the South African clawed frog,Xenopus laevis

A reproducible and effective method for fertilization eggs of Xenopus laevis was developed based of systematic manipulation of environmental factors. The effects of varying concentrations of individual components of a fertilization medium were tested by measuring jelly swelling, sperm motility, and sperm longevity. Results were used to develop an improved medium for fertilization, consisting of 41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl₂, 0.0625 mM MgCl₂, 0.5 mM Na₂HPO₄, 2.5 mM HEPES, 1.9 mM NaOH, final pH(2°) 7.8.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Xanthine dehydrogenase activity in the clawed frog, Xenopus laevis

Xanthine dehydrogenase (XDH; EC 1.2.1.37) activity in the clawed frog, Xenopus laevis, was detected in kidney tissue homogenates, but not in skin, liver, ovaries or gut tissues. The enzyme migrated as a single band of activity on both polyacrylamide and starch gel electropherograms, exhibited substrate inhibition, and did not appear developmentally until feeding larval stages. The tissue specificity, post-fertilization stage of appearance and single isozymic form make this a useful enzyme marker for further study concerning its developmental appearance and maintenance as a kidney-specific protein.     

Outgrowth dependency of forelimb regeneration on nerves in adult African clawed frog,Xenopus laevis

Summary The relationship between the size and shape of regenerative outgrowth and the quantity of innervation was studied in adult Xenopus laevis. The forelimbs, of which the nerve supply was artificially altered, were amputated midway through the stylopodium and were kept for 1 year. The regenerative outgrowths that formed in normal limbs with an intact nerve supply were mainly spike-shaped and occasionally rod-shaped. However, when the nerve supply to the distal part of the forelimb was augmented by surgically diverting ipsilateral sciatic nerve bundles, the quantity of innervation was increased to about two and a half times that of the normal limb. These hyperinnervated outgrowths were somewhat larger than those of the normally innervated outgrowths and the majority of them were oar-shaped, a type hardly ever encountered in normal regeneration. In contrast, when partial denervation was performed concomitantly with limb amputation, by ablation of the N. radialis at the shoulder joint, the quantity of innervation decreased to about one half that of the normal limb. The outgrowths obtained were spike-shaped in all cases, with their size being about half that of the normally innervated outgrowths. Furthermore, when both the N. radialis and N. ulnaris were ablated in the same way, the amputated limbs were mostly non-regenerative, but some of them regenerated small conical outgrowths. Based on these results, a discussion is presented concerning the relationship between a regenerative outgrowth and the innervation of the forelimb in Xenopus.     

Development of adrenergic and cholinergic cardiac control in larvae of the African clawed frog Xenopus laevis

Cardiac responses (heart rate, stroke volume, and cardiac output) to cholinergic and adrenergic receptor stimulation were investigated in developing larvae of Xenopus laevis from Nieuwkoop and Faber (NF) stage 33/34 (newly hatched) to NF stage 53 (22 d after hatching). Effects on heart rate (fH), stroke volume (SV), and cardiac output (CO) were analyzed using in situ preparations and video-microscopic techniques to record the continually beating heart. The results show that administration of acetylcholine to the heart decreases heart rate as early as NF stage 40. A significant reduction in SV and CO following acetylcholine administration to the heart was found at NF stages 45-53. Epinephrine had no significant effect on fH, SV, or CO at any of the stages investigated. However, an adrenergic tonus on the heart is present already at NF stage 40 (11%). This tonus increases up to a maximum (44%) at NF stages 45-47, when the maximal heart rate is found during development of X. laevis. We conclude that acetylcholine has a negative chronotropic and possibly also inotropic effect on the heart very early in development of X. laevis. We also hypothesize that the high adrenergic tonus found at NF stages 45-47 is responsible, at least in part, for the peak in heart rate seen at these stages.     

Biophysics of underwater hearing in the clawed frog,Xenopus laevis

Anesthetized clawed frogs (Xenopus laevis) were stimulated with underwater sound and the tympanic disk vibrations were studied using laser vibrometry. The tympanic disk velocities ranged from 0.01 to 0.5 mm/s (at a sound pressure of 2 Pa) in the frequency range of 0.4–4 kHz and were 20–40 dB higher than those of the surrounding tissue. The frequency response of the disk had two peaks, in the range of 0.6–1.1 kHz and 1.6–2.2 kHz, respectively. The first peak corresponded to the peak vibrations of the body wall overlying the lung. The second peak matched model predictions of the pulsations of the air bubble in the middle ear cavity. Filling the middle ear cavity with water lowered the disk vibrations by 10–30 dB in the frequency range of 0.5–3 kHz.Inflating the lungs shifted the low-frequency peak downwards, but did not change the high-frequency peak. Thus, the disk vibrations in the frequency range of the mating call (main energy at 1.7–1.9 kHz) were mainly caused by pulsations of the air in the middle ear cavity; sound transmission via the lungs was more important at low frequencies (below 1 kHz). Furthermore, the low-frequency peak could be reversibly reduced in amplitude by loading the larynx with metal or tissue glue. This shows that the sound-induced vibrations of the lungs are probably coupled to the middle ear cavities via the larynx. Also, anatomical observations show that the two middle ear cavities and the larynx are connected in an air-filled recess in submerged animals.This arrangement is unique to pipid frogs and may be a structural adaptation to connect all the air spaces of the frog and improve low-frequency underwater hearing. Another function of the recess may be to allow cross-talk between the two middle ear cavities. Thus, the ear might be directional. Our pilot experiments show up to 10 dB difference between ipsi- and contralateral stimulus directions in a narrow frequency range around 2 kHz.     

Ontogeny of hypoxic modulation of cardiac performance and its allometry in the African clawed frog Xenopus laevis

The ontogeny of cardiac hypoxic responses, and how such responses may be modified by rearing environment, are poorly understood in amphibians. In this study, cardiac performance was investigated in Xenopus laevis from 2 to 25 days post-fertilization (dpf). Larvae were reared under either normoxia or moderate hypoxia (PO₂ = 110 mmHg), and each population was assessed in both normoxia and acute hypoxia. Heart rate (f _h ) of normoxic-reared larvae exhibited an early increase from 77 ± 1 beats min^?1 at 2 dpf to 153 ± 1 beats min^?1 at 4 dpf, followed by gradual decreases to 123 ± 3 beats min^?1 at 25 dpf. Stroke volume (SV), 6 ± 1 nl, and cardiac output (CO), 0.8 ± 0.1 μl min^?1, at 5 dpf both increased by more than 40-fold to 25 dpf with rapid larval growth (~30-fold increase in body mass). When exposed to acute hypoxia, normoxic-reared larvae increased f _h and CO between 5 and 25 dpf. Increased SV in acute hypoxia, produced by increased end-diastolic volume (EDV), only occurred before 10 dpf. Hypoxic-reared larvae showed decreased acute hypoxic responses of EDV, SV and CO at 7 and 10 dpf. Over the period of 2–25 dpf, cardiac scaling with mass showed scaling coefficients of ?0.04 (f _h ), 1.23 (SV) and 1.19 (CO), contrary to the cardiac scaling relationships described in birds and mammals. In addition, f _h scaling in hypoxic-reared larvae was altered to a shallower slope of ?0.01. Collectively, these results indicate that acute cardiac hypoxic responses develop before 5 dpf. Chronic hypoxia at a moderate level can not only modulate this cardiac reflex, but also changes cardiac scaling relationship with mass.     

Tradeoffs between somatic and gonadal investments during development in the African clawed frog (Xenopus laevis)

Tradeoffs between time to and size at metamorphosis occur in many organisms with complex life histories. The ability to accelerate metamorphosis can increase survival to the next life stage, but the resulting smaller size at metamorphosis is often associated with lower post-metamorphic survival or reduced fecundity of adults. Reduced fecundity is thought to be because of reduced energy reserves, longer time to maturity, or reduced capacity to carry eggs or compete for mates. This pattern could also be explained by a shift in allocation to somatic growth that further retards the growth or development of reproductive tissues. The main goal of this study was to determine if the relationship between growth and development of somatic and gonadal tissues depends on environmental conditions. We address this question through two experiments in which we quantify the development and growth of the body and gonads of Xenopus laevis reared in different resource environments. First, tadpoles were reared communally and development and growth were evaluated over time. Restricted food reduced somatic and gonadal growth rate, but did not affect the developmental rate of either tissue type. Second, tadpoles were reared individually and evaluated at metamorphosis. Restricted food reduced somatic development and growth, but only influenced size, and not developmental stage of testes at metamorphosis. This work demonstrates that environmental conditions influence tradeoffs between growth and development of somatic and gonadal tissues, apparently in a sex-specific manner. These tradeoffs may contribute to phenotypic correlations between small size and reduced fitness.     

Low genetic diversity of the successful invasive African clawed frog Xenopus laevis (Pipidae) in Chile

In Africa, the genus Xenopus presents cryptic species and diverse hybrids between species. It has been assumed that the invasive populations of this genus correspond to X. laevis and that they are derived from the subspecies that inhabits the Mediterranean Cape region of South Africa. In part, this is supported by the successful establishment of this species in several Mediterranean regions of the world. In Mediterranean Chile, Xenopus has invaded an area of about 21,000 km², with scarce attention to genetic aspects underlying its invasion. Using mitochondrial DNA sequences we determined that Xenopus laevis laevis from the Cape region of South Africa is the subspecies that invaded Chile. The analysis indicated that the invaders have low genetic diversity (only two haplotypes, compared to 10 in two localities of their native range), and that probably the invasion in Chile occurred only once. Landscape genetics revealed that factors such as aridity and elevation have determined the spread of the species, both from the ecological and genetic points of view. Our results show that the invasion of the African clawed frog in Chile has been successful for at least 30 years, in spite of low genetic variability, few events of introduction, low propagule pressure, and bottlenecks in the founding population.     

Ontogeny of excitatory and inhibitory control of gastrointestinal motility in the African clawed frog, Xenopus laevis

The transparent body wall of Xenopus laevis larvae during the first developmental stages allows in vivo studies of gastrointestinal tract activity. The purpose of this study was to chart the ontogeny of gut motility in Xenopus larvae and to identify the most important control systems during the first developmental stages. Coordinated descending contraction waves first occurred in the gut at Nieuwkoop and Faber stage 43 [0.8 +/- 0.1 contractions/min (cpm)] and increased to 4.9 +/- 0.1 cpm at stage 47. The cholinergic receptor agonist carbachol (5-10 microM) increased contraction frequency already at stage 43, as did neurokinin A (NKA, 0.3-1 microM). The muscarinic antagonist atropine (100 microM) first affected contraction frequency at stage 45, which coincides with the onset of feeding. The tachykinin antagonist MEN-10,376 (6 microM) blocked NKA-induced contractions but not spontaneous motility. Both sodium nitroprusside [nitric oxide (NO) donor, 1-10 microM] and vasoactive intestinal peptide (VIP, 0.1-1 microM) inhibited contractions from the earliest stage onward. Blocking NO synthesis using NG-nitro-L-arginine methyl ester (100 microM) had no effect per se, but antagonized VIP evoked inhibition at stage 47. We conclude that gastrointestinal motility is well developed in the Xenopus laevis larvae before the onset of feeding. Functional muscarinic and tachykinin receptors are present already at the onset of motility, whereas a cholinergic tone develops around the onset of feeding. No endogenous tachykinin tone was found. Functional VIP receptors mediate inhibition at the onset of motility. NO seems to mediate the VIP effect at later stages.     

Genetic basis of spectral tuning in the violet-sensitive visual pigment of African clawed frog, Xenopus laevis

Ultraviolet (UV) and violet vision in vertebrates is mediated by UV and violet visual pigments that absorb light maximally (lambdamax) at approximately 360 and 390-440 nm, respectively. So far, a total of 11 amino acid sites only in transmembrane (TM) helices I-III are known to be involved in the functional differentiation of these short wavelength-sensitive type 1 (SWS1) pigments. Here, we have constructed chimeric pigments between the violet pigment of African clawed frog (Xenopus laevis) and its ancestral UV pigment. The results show that not only are the absorption spectra of these pigments modulated strongly by amino acids in TM I-VII, but also, for unknown reasons, the overall effect of amino acid changes in TM IV-VII on the lambdamax-shift is abolished. The spectral tuning of the contemporary frog pigment is explained by amino acid replacements F86M, V91I, T93P, V109A, E113D, L116V, and S118T, in which V91I and V109A are previously unknown, increasing the total number of critical amino acid sites that are involved in the spectral tuning of SWS1 pigments in vertebrates to 13.     

Realized niche shift associated with Galinsoga quadriradiata (Asteraceae) invasion in China

粗毛牛膝菊在中国的入侵与生态位漂移有关 在外来物种入侵和扩散过程中,生态位的漂移可能起到了重要作用。粗毛牛膝菊(Galinsoga quadriradiata) 在中国已造成了较为严重的入侵,占据了许多与其原产地不同的气候区。为此,本研究力图揭示粗毛牛膝菊入侵过程中的气候生态位漂移,分析其在该物种入 侵中国过程中可能发挥的作用。本研究结合粗毛牛膝菊原 产地和入侵地的分布点与气候数据, 采用Maxent模型预测了其在中国潜在的分布,并采用主成分分析的方法评估 了在入侵中国过程中粗毛牛膝菊气候生态位的漂移。模型结果显示,该物种原产地种群和入侵地种群之间只 有32.7%的生态位重叠,两个种群的生态位相似性较低(Schoener's D = 0.093, P < 0.005),这暗示了在其入侵过程中发生了生态位漂移。相比于其原产地种群,其在中国的入侵种群气候生态位的整体范围和中心都明 显地漂移向了温度更低、降水更少的区域;中国南方大部分区域属于粗毛牛膝菊的稳定适生区,而位于入侵 前沿的北方地区则存在局域适应和潜在拓殖区域。这些研究结果说明,粗毛牛膝菊在中国的入侵种群仍处于准平衡阶段,未来有可能继续向新的适生区扩散入侵,其生态位的变化有力地解释了为什么该物种在中国的入侵性强、危害范围大。     

Differential antigen expression during metamorphosis in the tripartite olfactory system of the African clawed frog, Xenopus laevis

In the adult African clawed frog, Xenopus laevis, olfactory epithelium is housed in three separate nasal cavities: the principal cavity, the middle cavity, and the vomeronasal organ. The sensory epithelium in each of these cavities has distinct cellular features, and presumed physiological and behavioral functions, which arise during metamorphosis. Most notably, the middle cavity is formed de novo, and the principal cavity is transformed from a larval sensory epithelium with water exposure to an adult olfactory epithelium with air exposure. To understand the cellular nature of this plasticity more clearly, we characterized the staining patterns generated in the olfactory system of X. laevis with a new monoclonal antibody, anti-E7. The olfactory epithelium is first stained with anti-E7 during late embryonic development. Transection of the olfactory nerves during metamorphosis eliminates all staining and indicates that the staining is associated with mature or nearly mature olfactory receptor neurons. The antibody diffusely stains the vomeronasal organ throughout development and in adults. In the larval principal cavity, the olfactory receptor neurons are brightly stained, but this cellular staining is lost after metamorphosis. The mucus from Bowman's glands in the principal cavity, however, is intensely stained in adults. The middle cavity, throughout development and in adulthood, has the same staining characteristics as the larval principal cavity. Thus, the E7 antibody can distinguish the three areas of the olfactory epithelium, allowing measurement of sensory epithelium volume, and serves as an excellent marker for the changes in the sensory epithelium that occur during metamorphosis.